Indacaterol inhibits collective cell migration and IGDQ-mediated single cell migration in metastatic breast cancer MDA-MB-231 cells.

Metastasis is the main cause of deaths related to breast cancer. This is particular the case for triple negative breast cancer. No targeted therapies are reported as efficient until now. The extracellular matrix, in particular the fibronectin type I motif IGDQ, plays a major role in regulating cell migration prior metastasis formation. This motif interacts with specific integrins inducing their activation and the migratory signal transduction.Here, we characterized the migratory phenotype of MDA-MB-231 cells, using functionalized IGDQ-exposing surfaces, and compared it to integrin A5 and integrin B3 knock-down cells. A multiomic analysis was developed that highlighted the splicing factor SRSF6 as a putative master regulator of cell migration and of integrin intracellular trafficking. Indacaterol-induced inhibition of SRSF6 provoked: i) the inhibition of collective and IGDQ-mediated cell migration and ii) ITGA5 sequestration into endosomes and lysosomes. Upon further studies, indacaterol may be a potential therapy to prevent cell migration and reduce metastasis formation in breast cancer. Video Abstract.

Priming of mesenchymal stem cells with a hydrosoluble form of curcumin allows keeping their mesenchymal properties for cell-based therapy development.

Mesenchymal stem cells are increasingly studied for their use as drug-carrier in addition to their intrinsic potential for regenerative medicine. They could be used to transport molecules with a poor bioavailability such as curcumin in order to improve their clinical usage. This natural polyphenol, well-known for its antioxidant and anti-inflammatory properties, has a poor solubility that limits its clinical potential. For this purpose, the use of NDS27, a curcumin salt complexed with hydroxypropyl-beta-cyclodextrin (HPßCD), displaying an increased solubility in aqueous solution, is preferred. This study aims to evaluate the uptake of NDS27 into skeletal muscle-derived mesenchymal stem cells (mdMSCs) and the effects of such uptake onto their mesenchymal properties. It appeared that the uptake of NDS27 into mdMSCs is concentration-dependent and not time-dependent. The use of a concentration of 7 µmol/L which does not affect the viability and proliferation also allows preservation of their adhesion, invasion and T cell immunomodulatory abilities.

The Transcription Factor 7-Like 2-Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1 Alpha Axis Connects Mitochondrial Biogenesis and Metabolic Shift with Stem Cell Commitment to Hepatic Differentiation.

Increasing evidence supports that modifications in the mitochondrial content, oxidative phosphorylation (OXPHOS) activity, and cell metabolism influence the fate of stem cells. However, the regulators involved in the crosstalk between mitochondria and stem cell fate remains poorly characterized. Here, we identified a transcriptional regulatory axis, composed of transcription factor 7-like 2 (TCF7L2) (a downstream effector of the Wnt/ß-catenin pathway, repressed during differentiation) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) (the master regulator of mitochondrial biogenesis, induced during differentiation), coupling the loss of pluripotency and early commitment to differentiation, to the initiation of mitochondrial biogenesis and metabolic shift toward OXPHOS. PGC-1α induction during differentiation is required for both mitochondrial biogenesis and commitment to the hepatocytic lineage, and TCF7L2 repression is sufficient to increase PGC-1α expression, mitochondrial biogenesis and OXPHOS activity. We further demonstrate that OXPHOS activity is required for the differentiation toward the hepatocytic lineage, thus providing evidence that bi-directional interactions control stem cell differentiation and mitochondrial abundance and activity. Stem Cells 2017;35:2184-2197.

Proteomic Study of SUMOylation During Solanum tuberosum-Phytophthora infestans Interactions.

Invasive plant pathogens have developed the ability to modify the metabolism of their host, promoting metabolic processes that facilitate the growth of the pathogen at the general expense of the host. The particular enzymatic process SUMOylation, which performs posttranslational modification of target proteins, leading to changes in many aspects of protein activity and, hence, metabolism, has been demonstrated to be active in many eukaryotic organisms, both animals and plants. Here, we provide experimental evidence that indicates that, in leaves of Solanum tuberosum that have been infected by Phytophthora infestans, the SUMO (small ubiquitin-like modifier) pathway enzymes of the host are partially under transcriptional control exerted by the oomycete. Using a recently developed approach that employs three-dimensional gels, we show that, during the infection process, the abundances of most of the known SUMO conjugates of S. tuberosum change significantly, some decreasing, but many increasing in abundance. The new proteomic approach has the potential to greatly facilitate investigation of the molecular events that take place during the invasion by a pathogen of its host plant.

Effects of a Sublethal and Transient Stress of the Endoplasmic Reticulum on the Mitochondrial Population.

Endoplasmic reticulum (ER) and mitochondria are not discrete intracellular organelles but establish close physical and functional interactions involved in several biological processes including mitochondrial bioenergetics, calcium homeostasis, lipid synthesis, and the regulation of apoptotic cell death pathways. As many cell types might face a transient and sublethal ER stress during their lifetime, it is thus likely that the adaptive UPR response might affect the mitochondrial population. The aim of this work was to study the putative effects of a non-lethal and transient endoplasmic reticulum stress on the mitochondrial population in HepG2 cells. The results show that thapsigargin and brefeldin A, used to induce a transient and sublethal ER stress, rapidly lead to the fragmentation of the mitochondrial network associated with a decrease in mitochondrial membrane potential, O2 (•-) production and less efficient respiration. These changes in mitochondrial function are transient and preceded by the phosphorylation of JNK. Inhibition of JNK activation by SP600125 prevents the decrease in O2 (•-) production and the mitochondrial network fragmentation observed in cells exposed to the ER stress but has no impact on the reduction of the mitochondrial membrane potential. In conclusion, our data show that a non-lethal and transient ER stress triggers a rapid activation of JNK without inducing apoptosis, leading to the fragmentation of the mitochondrial network and a reduction of O2 (•-) production. J. Cell. Physiol. 231: 1913-1931, 2016. © 2015 Wiley Periodicals, Inc.

New Pathophysiological Insights from Serum Proteome Profiling in Equine Atypical Myopathy.

Equine atypical myopathy (AM) is a severe environmental intoxication linked to the ingestion of protoxins contained in seeds and seedlings of the sycamore maple (Acer pseudoplatanus) in Europe. The toxic metabolites cause a frequently fatal rhabdomyolysis syndrome in grazing horses. Since these toxic metabolites can also be present in cograzing horses, it is still unclear as to why, in a similar environmental context, some horses show signs of AM, whereas others remain clinically healthy. Label-free proteomic analyses on the serum of 26 diseased AM, 23 cograzers, and 11 control horses were performed to provide insights into biological processes and pathways. A total of 43 and 44 differentially abundant proteins between "AM vs cograzing horses" and "AM vs control horses" were found. Disease-linked changes in the proteome of different groups were found to correlate with detected amounts of toxins, and principal component analyses were performed to identify the 29 proteins representing a robust AM signature. Among the pathway-specific changes, the glycolysis/gluconeogenesis pathway, the coagulation/complement cascade, and the biosynthesis of amino acids were affected. Sycamore maple poisoning results in a combination of inflammation, oxidative stress, and impaired lipid metabolism, which is trying to be counteracted by enhanced glycolysis.

Characterization of the Proteins Secreted by Equine Muscle-Derived Mesenchymal Stem Cells Exposed to Cartilage Explants in Osteoarthritis Model.

BACKGROUND: Osteoarthritis (OA) is a highly prevalent joint degenerative disease for which therapeutic treatments are limited or invasive. Cell therapy based on mesenchymal stem/stromal cells (MSCs) is therefore seen as a promising approach for this disease, in both human and horses. As the regenerative potential of MSCs is mainly conferred by paracrine function, the goal of this study was to characterize the secreted proteins of muscle-derived MSCs (mdMSCs) in an in vitro model of OA to evaluate the putative clinical interest of mdMSCs as cell therapy for joint diseases like osteoarthritis. METHODS: An equine osteoarthritis model composed of cartilage explants exposed to pro-inflammatory cytokines was first developed. Then, the effects of mdMSC co-culture on cartilage explant were studied by measuring the glycosaminoglycan release and the NO2- production. To identify the underlying molecular actors, stable isotope-labeling by amino acids in cell culture based secreted protein analyses were conducted, in the presence of serum. The relative abundance of highly sequenced proteins was finally confirmed by western blot. RESULTS: Co-culture with muscle-derived MSCs decreases the cytokine-induced glycosaminoglycan release by cartilage explants, suggesting a protecting effect of mdMSCs. Among the 52 equine proteins sequenced in the co-culture conditioned medium, the abundance of decorin and matrix metalloproteinase 3 was significantly modified, as confirmed by western blot analyses. CONCLUSIONS: These results suggest that muscle-derived MSCs could reduce the catabolic effect of TNFα and IL-1ß on cartilage explant by decreasing the secretion and activity of matrix metalloproteinase 3 and increasing the decorin secretion. mdMSCs capacity to reduce the catabolic consequences of cartilage exposure to pro-inflammatory cytokines. These effects can be explained by mdMSC-secreted bioactive such as TIMP-1 and decorin, known as an inhibitor of MMP3 and an anti-inflammatory protein, respectively.

The global downregulation of protein synthesis observed during hepatogenic maturation is associated with a decrease in TOP mRNA translation.

Translational regulation is of paramount importance for proteome remodeling during stem cell differentiation at both the global and the transcript-specific levels. In this study, we characterized translational remodeling during hepatogenic differentiation of induced pluripotent stem cells (iPSCs) by polysome profiling. We demonstrate that protein synthesis increases during exit from pluripotency and is then globally repressed during later steps of hepatogenic maturation. This global downregulation of translation is accompanied by a decrease in the abundance of protein components of the translation machinery, which involves a global reduction in translational efficiency of terminal oligopyrimidine tract (TOP) mRNA encoding translation-related factors. Despite global translational repression during hepatogenic differentiation, key hepatogenic genes remain efficiently translated, and the translation of several transcripts involved in hepatospecific functions and metabolic maturation is even induced. We conclude that, during hepatogenic differentiation, a global decrease in protein synthesis is accompanied by a specific translational rewiring of hepatospecific transcripts.

Senescence Induced by UVB in Keratinocytes Impairs Amino Acids Balance.

Skin is one of the most exposed organs to external stress. Namely, UV rays are the most harmful stress that could induce important damage leading to skin aging and cancers. At the cellular level, senescence is observed in several skin cell types and contributes to skin aging. However, the origin of skin senescent cells is still unclear but is probably related to exposure to stresses. In this work, we developed an in vitro model of UVB-induced premature senescence in normal human epidermal keratinocytes. UVB-induced senescent keratinocytes display a common senescent phenotype resulting in an irreversible cell cycle arrest, an increase in the proportion of senescence-associated ß-galactosidase‒positive cells, unrepaired DNA damage, and a long-term DNA damage response activation. Moreover, UVB-induced senescent keratinocytes secrete senescence-associated secretory phenotype factors that influence cutaneous squamous cell carcinoma cell migration. Finally, a global transcriptomic study highlighted that senescent keratinocytes present a decrease in the expression of several amino acid transporters, which is associated with reduced intracellular levels of glycine, alanine, and leucine. Interestingly, the chemical inhibition of the glycine transporter SLC6A9/Glyt1 triggers senescence features.

Taking Advantage of the Senescence-Promoting Effect of Olaparib after X-ray and Proton Irradiation Using the Senolytic Drug, ABT-263.

Radiotherapy (RT) is a key component of cancer treatment. Although improvements have been made over the years, radioresistance remains a challenge. For this reason, a better understanding of cell fates in response to RT could improve therapeutic options to enhance cell death and reduce adverse effects. Here, we showed that combining RT (photons and protons) to noncytotoxic concentration of PARP inhibitor, Olaparib, induced a cell line-dependent senescence-like phenotype. The senescent cells were characterized by morphological changes, an increase in p21 mRNA expression as well as an increase in senescence-associated ß-galactosidase activity. We demonstrated that these senescent cells could be specifically targeted by Navitoclax (ABT-263), a Bcl-2 family inhibitor. This senolytic drug led to significant cell death when combined with RT and Olaparib, while limited cytotoxicity was observed when used alone. These results demonstrate that a combination of RT with PARP inhibition and senolytics could be a promising therapeutic approach for cancer patients.

IGDQ motogenic peptide gradient induces directional cell migration through integrin (αv)ß3 activation in MDA-MB-231 metastatic breast cancer cells.

In the context of breast cancer metastasis study, we have shown in an in vitro model of cell migration that IGDQ-exposing (IsoLeu-Gly-Asp-Glutamine type I Fibronectin motif) monolayers (SAMs) on gold sustain the adhesion of breast cancer MDA-MB-231 cells by triggering Focal Adhesion Kinase and integrin activation. Such tunable scaffolds are used to mimic the tumor extracellular environment, inducing and controlling cell migration. The observed migratory behavior induced by the IGDQ-bearing peptide gradient along the surface allows to separate cell subpopulations with a "stationary" or "migratory" phenotype. In this work, we knocked down the integrins α5(ß1) and (αv)ß since they are already known to be implicated in cell migration. To this aim, a whole proteomic analysis was performed in beta 3 integrin (ITGB3) or alpha 5 integrin (ITGA5) knock-down MDA-MB-231 cells, in order to highlight the pathways implied in the integrin-dependent cell migration. Our results showed that i) ITGB3 depletion influenced ITGA5 mRNA expression, ii) ITGB3 and ITGA5 were both necessary for IGDQ-mediated directional single cell migration and iii) integrin (αv)ß3 was activated by IGDQ fibronectin type I motif. Finally, the proteomic analysis suggested that co-regulation of recycling transport of ITGB3 by ITGA5 is potentially necessary for directional IGDQ-mediated cell migration.

DNA repair during nonreductional meiosis in the asexual rotifer Adineta vaga.

Rotifers of the class Bdelloidea are microscopic animals notorious for their long-term persistence in the apparent absence of sexual reproduction and meiotic recombination. This evolutionary paradox is often counterbalanced by invoking their ability to repair environmentally induced genome breakage. By studying the dynamics of DNA damage response in the bdelloid species Adineta vaga, we found that it occurs rapidly in the soma, producing a partially reassembled genome. By contrast, germline DNA repair is delayed to a specific time window of oogenesis during which homologous chromosomes adopt a meiotic-like juxtaposed configuration, resulting in accurate reconstitution of the genome in the offspring. Our finding that a noncanonical meiosis is the mechanism of germline DNA repair in bdelloid rotifers gives previously unidentified insights on their enigmatic long-term evolution.

Hypersensitivity of mtDNA-depleted cells to staurosporine-induced apoptosis: roles of Bcl-2 downregulation and cathepsin B.

We show that mitochondrial DNA (mtDNA)-depleted 143B cells are hypersensitive to staurosporine-induced cell death as evidenced by a more pronounced DNA fragmentation, a stronger activation of caspase-3, an enhanced poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, and a more dramatic cytosolic release of cytochrome c. We also show that B-cell CLL/lymphoma-2 (Bcl-2), B-cell lymphoma extra large (Bcl-X(L)), and myeloid cell leukemia-1 (Mcl-1) are constitutively less abundant in mtDNA-depleted cells, that the inhibition of Bcl-2 and Bcl-X(L) can sensitize the parental cell line to staurosporine-induced apoptosis, and that overexpression of Bcl-2 or Bcl-X(L) can prevent the activation of caspase-3 in ρ(0)143B cells treated with staurosporine. Moreover, the inactivation of cathepsin B with CA074-Me significantly reduced cytochrome c release, caspase-3 activation, PARP-1 cleavage, and DNA fragmentation in mtDNA-depleted cells, whereas the pan-caspase inhibitor failed to completely prevent PARP-1 cleavage and DNA fragmentation in these cells, suggesting that caspase-independent mechanisms are responsible for cell death even if caspases are activated. Finally, we show that cathepsin B is released in the cytosol of ρ(0) cells in response to staurosporine, suggesting that the absence of mitochondrial activity leads to a facilitated permeabilization of lysosomal membranes in response to staurosporine.

Identification of DH IC-2 as a HIF-1 independent protein involved in the adaptive response to hypoxia in tumor cells: A putative role in metastasis.

The master regulator of the adaptive response to hypoxia is HIF-1. However, while some data show that HIF-1 can control more than 80% of the genes induced under hypoxia, other experiments clearly demonstrate that a part of the hypoxic response is independent of HIF-1. The goal of this study was to identify some of these HIF-1 independent factors and to investigate their functional role in the adaptation of tumor cells to hypoxia. We show that the cytoplasmic dynein intermediate chain 2 (DH IC-2), a component of an intracellular ATPase minus-end directed tubulin-based motile complex, was stabilized and post-translationally modified under hypoxia in a HIF-1 independent way. We identified this modification as a phosphorylation by protein kinase C, which is inhibited under hypoxia. In parallel, the migration of HepG2 cells was enhanced under hypoxia. Cell migration was also increased, to the same extent, by the invalidation of DH IC-2 using siRNA. Taken together, these results suggest that under hypoxia, a specific modification of DH IC-2 may modulate its activity, and in turn promote cell migration. These results are important to better understand cancer development since they highlight a HIF-1 independent mechanism, which may be involved in metastasis.

Repeated exposures to UVB induce differentiation rather than senescence of human keratinocytes lacking p16(INK-4A).

Skin cancers and extrinsic aging are delayed consequences of cumulative UV radiation insults. Exposure of human keratinocytes to UVB has been previously shown to trigger premature senescence. In order to explore the involvement of the cyclin-dependent kinase inhibitor p16(INK-4a) in UVB-induced premature senescence, we developed an original model of repeated sublethal exposures of human keratinocytes deficient in p16(INK-4a). We did not observe any significant increase of senescence-associated beta-galactosidase activity positive cells following UVB exposure in this cell line in contrast to primary keratinocytes, suggesting a role for p16(INK-4a) in UVB-induced senescence. However, we detected sustained DNA damage, prolonged cell cycle arrest, and induction of markers of epidermal differentiation like involucrin and filaggrin as consequences of the repeated exposures. Keratinocytes exposed to the same dose of UVB in a single exposure died. Furthermore, the abundance of the keratins 6, 16 and 17 was increased in keratinocytes exposed repeatedly to UVB suggesting an alternative differentiation. This model allows the induction of a state of differentiation observed in vivo with differentiation uncoupled from premature senescence.

Cycling hypoxia promotes a pro-inflammatory phenotype in macrophages via JNK/p65 signaling pathway.

Cycling hypoxia (cyH), also called intermittent hypoxia, occurs in solid tumors and affects different cell types in the tumor microenvironment and in particular the tumor-associated macrophages (TAMs). As cyH and TAMs both favor tumor progression, we investigated whether cyH could drive the pro-tumoral phenotype of macrophages. Here, the effects of cyH on human THP-1 macrophages and murine bone marrow-derived macrophages (BMDM), either unpolarized M0, or polarized in M1 or M2 phenotype were studied. In M0 macrophages, cyH induced a pro-inflammatory phenotype characterized by an increase in TNFα and IL-8/MIP-2 secretion. CyH amplified the pro-inflammatory phenotype of M1 macrophages evidenced by an increased pro-inflammatory cytokine secretion and pro-inflammatory gene expression. Furthermore, cyH increased c-jun activation in human M0 macrophages and highly increased c-jun and NF-κB activation in M1 macrophages. C-jun and p65 are implicated in the effects of cyH on M0 and M1 macrophages since inhibition of their activation prevented the cyH pro-inflammatory effects. In conclusion, we demonstrated that cyH induces or amplifies a pro-inflammatory phenotype in M0 and M1 macrophages by activating JNK/p65 signaling pathway. These results highlight a specific role of cyH in the amplification of tumor-related inflammation by modulating the inflammatory phenotype of macrophages.

Optimization of label-free nano LC-MS/MS analysis of the placental proteome.

The placenta can be regarded as a mirror of the events to which the fetus is exposed during development. The placental proteome has been studied with several methodologies differing in sample handling, protein extraction, and processing. We optimized a protocol to analyze the placental proteome by means of label-free nano-LC-MS/MS mass spectrometry with regard to sample treatment, protein extraction, and protein digestion, in order to obtain a high protein concentration for identification of a specific protein signature according to the conditions studied. We recommend mechanical tissue disruption, blood removal prior to protein extraction, and FASP-based or in-gel digestion.

Characterization of the role of TMEM45A in cancer cell sensitivity to cisplatin.

TMEM45A is a transmembrane protein involved in tumor progression and cancer resistance to chemotherapeutic agents in hypoxic condition. It is correlated to a low breast cancer patient overall survival. However, little is known about this protein, in particular the mechanisms by which TMEM45A modulates cancer cell chemosensitivity. In this work, the messenger RNA expression of TMEM45A was assessed in head and neck squamous cell carcinoma (HNSCC) and renal cell carcinoma (RCC) biopsies. TMEM45A was upregulated in patients diagnosed for head and neck or renal cancer. Then, the implication of this protein in cisplatin sensitivity was explored in SQD9 and RCC4 + pVHL cells. TMEM45A inactivation decreased cell proliferation and modulated cell responses to cisplatin. Indeed, TMEM45A inactivation increased the sensitivity of SQD9 cells to cisplatin, whereas it rendered RCC4 + pVHL cells resistant to this anticancer agent. Through RNA-sequencing analysis, we identified several deregulated pathways that indicated that the impact on cisplatin sensitivity may be associated to the inhibition of DNA damage repair and to UPR pathway activation. This study demonstrated, for the first time, an anti or a pro-apoptotic role of this protein depending on the cancer type and highlighted the role of TMEM45A in modulating patient responses to treatment.

Increased abundance of cytoplasmic and nuclear caveolin 1 in human diploid fibroblasts in H(2)O(2)-induced premature senescence and interplay with p38alpha(MAPK).

Treatment of IMR-90 human diploid fibroblasts with a sublethal concentration of H(2)O(2) induces premature senescence. We investigated the protein abundance, subcellular localization and involvement of caveolin 1 in premature senescence. Caveolin 1 is a scaffolding protein able to concentrate and organize signaling molecules within the caveolae membrane domains. We report the first evidence of increased nuclear and cytoplasmic localization of caveolin 1 during establishment of H(2)O(2)-induced premature senescence. Moreover, we demonstrate that phosphorylation of caveolin 1 during treatment with H(2)O(2) is dependent on p38alpha mitogen-activated protein kinase.

Upregulation of annexin A2 in H(2)O(2)-induced premature senescence as evidenced by 2D-DIGE proteome analysis.

Human diploid fibroblasts undergo premature senescence after treatment with sublethal concentration of H(2)O(2). We report the first proteomic study of microsomal proteins in the context of H(2)O(2)-induced premature senescence by using 2D-DIGE approach. Twelve different proteins with altered abundance at day 3 after treatment with H(2)O(2) were identified. Among them, we demonstrated a re-localization of annexin A2 in plasma membrane.